This project has successfully led to the development of the first in vitro model system in which the biologic behavior of dormant or proliferative metastatic cells can be correlated to their growth properties in our in vitro system. This has important implications in the field since now it is possible to study how the extracellular matrix (ECM) influences the transition from quiescent to proliferative growth. Additionally, we have reported the development of an in vivo model system where dormant tumor cells can be triggered to proliferate in a fibrotic, metastatic site. This provides an important new model system in which to investigate in vivo how changes in the ECM influence tumor cell dormancy and that can be used to test therapies that may inhibit the dormant-to-proliferative switch induced by fibrosis. We have demonstrated that the composition of the matrix can determine whether dormant cells remain quiescent. Our studies have shown that at least two components of the extracellular matrix, fibronectin and collagen type 1 can activate the dormant cells to proliferate through the engagement of integrin beta 1, a key membrane protein that mediates signaling from the extracellular matrix to the cell nucleus leading to profound molecular changes. We have further demonstrated that collagen I often found in tumors with poor prognosis or a high propensity to metastasize can activate the same pathway. Our molecular studies have dissected this molecular pathway in detail and we have identified potential targets that may inhibit this process. Recently, we have demonstrated using this model that the in vivo dormant-to-proliferative switch can be prevented through pharmacologic manipulation. We continue to explore how this therapy can be enhanced using combination therapy approaches. It is possible that these findings can be translated into more effective ways of preventing late-stage cancer recurrence. This work continues in parallel with our studies focused on understanding signaling pathways that are critical for initiating a proliferative response from the dormant tumor cells. A highly novel finding of this work was that the dormant to proliferative switch requires a major change in the architecture of the cytoskeleton (Barkan, Cancer research, 2008 and Barkan et al, Cancer Research,2010). If this process of actin stress fiber is inhibited by several methods, the cells remain dormant. This provides an additional set of molecular targets to test as inhibitors of the dormant-to-proliferative switch that may reduce the risk of tumor recurrence. We are testing new novel model systems in which we are able to alter the tumor microenvironment in mice to determine how such changes affect the growth of dormant tumor cells. Since changes in the microenvironment are associated with significant increases in risk of breast cancer mortality and tumor recurrence, these studies will provide very relevant models to study these processes that cannot be studied in human patients. The study of tumor cell dormancy is an area that has been minimally explored but accounts for a major cause of death from recurrent cancer. We have recently demonstrated that the combination of a Src and a Mek1/2 inhibitor inhibited the outbreak of dormant cells to proliferative metastatic lesions both in vitro and in vivo and led to apoptosis of dormant cells. This may have translational value. Therefore, these studies will provide important insights into this area that is highly relevant to the mission of NCI.